Platelet-derived growth factor (PDGF), the primary mitogen in serum for mesenchymal-derived cells, is stored in platelet alpha-granules. Injury to blood vessels activates the release of PDGF from these granules in the vicinity of the injured vessels. This mitogen acts as a potent chemoattractant for fibroblasts and smooth muscle cells, as well as monocytes and neutrophils. The mitogenic activity of the localized PDGF results in proliferation of these cells at the site of injury, contributing to the process of wound repair.
Purified native platelet-derived growth factor (PDGF), a glycoprotein of about 30,000 daltons, is composed of two disulfide-linked polypeptide chains. Two forms of these chains, designated A and B, have been identified. The native protein occurs as the homodimer AA or BB or the heterodimer AB, or a mixture thereof. A partial amino acid sequence for the PDGF-A chain has been identified (Johnsson et al. (1984) EMBO J. 3:921-928) and cDNAs encoding two forms of PDGF A-chain precursors have been described (U.S. Pat. No. 5,219,759). The mature A-chain consists of a 104 amino acid polypeptide that is derived by proteolytic processing of a 211 amino acid precursor polypeptide. The cDNA encoding the PDGF-B chain has also been described (Nature (1985) 316:748-750). The mature B-chain consists of a 109 amino acid polypeptide that is derived by proteolytic processing of a 241 amino acid precursor polypeptide. The mature A and B chains of PDGF show sequence identity of 51%, with the eight cysteine residues being conserved in each of the chains (Johnsson et al. (1984) EMBO J. 3:921-928).
In addition to proteolytic processing of the precursor polypeptides into mature PDGF-A and PDGF-B chains, recombinant PDGF (rPDGF) produced in a yeast host undergoes further post-translational processing that results in a secreted mature protein having considerable structural heterogeneity. This is true for the heterodimer rPDGF-AB, and more particularly for the homodimer rPDGF-BB.
When expressed in a yeast host for production of a bulk drug substance, PDGF undergoes endoproteolytic cleavage, or so-called clipping, of the B-chain between the Arg-32 and Thr-33 residues, resulting in a bulk drug substance having a mixture of unclipped or so-called intact rPDGF and clipped rPDGF that has been cleaved in the B-chain between the Arg-32 and Thr-33 residues. In the case of rPDGF-BB, one or both of the B-chains may be clipped, resulting in single-clipped or double-clipped rPDGF-BB, respectively. Other post-translational modifications of interest to the present invention include exoproteolytic removal of C-terminal amino acids, or so-called truncation, which may remove Arg-32 from clipped B-chains and/or Thr-109 from intact and clipped B-chains. These post-translational modifications lead to a number of structural forms, or so-called isoforms, of rPDGF-BB present in the secreted product. Methods of the present invention are directed to separation and purification of the intact, single-clipped, and double-clipped isoforms of rPDGF, more particularly rPDGF-BB.
The three dimeric forms of PDGF exhibit different binding affinities for the two known PDGF receptor gene products, .alpha. and .beta.. The beta receptor recognizes PDGF B chain and is dimerized in the presence of PDGF-BB. The alpha receptor recognizes PDGF B and A chains and can be dimerized by PDGF-BB, PDGF-AA, and PDGF-AB (see, for example, Abboud et al (1994) J. Cell. Phys. 158:140-150). The amino acid residue region of PDGF-BB involved in binding or activation of the receptor has been narrowed down to residues Ile25-Phe37 (Giese et al. (1990) Mol. Cell. Biol. 10:5496-5501). These residues include the Arg-32/Thr-33 site cleaved by endoproteolytic processing during production of PDGF-BB by a yeast host
Proteinase-resistant mutants of recombinant PDGF-BB exhibiting improved biological activity have been identified (Cook et al. (1992) Biochem. J. 281:57-65; see also European Patent Application No. 0 547 064 B1). These mutants prevent endoproteolytic cleavage of the B-chains at Arg-32. Thus elimination of endoproteolytic cleavage in this region apparently leads to increased biological activity of the PDGF-BB produced in a yeast host during the fermentation process. This increase in biological activity is presumably associated with an increase in the relative amount of unclipped, or so-called intact, PDGF-BB and a decrease in the relative amount of clipped PDGF-BB in the recombinant PDGF-BB product Similarly, endoproteolytic cleavage in this region of either or both of the B-chains within a rPDGF-BB protein would be expected to yield a bulk drug substance having different biological activity than found for a bulk drug substance consisting solely of the intact rPDGF-BB.
Prior to the present invention, methods commonly used in the art to purify recombinantly produced PDGF have not distinguished between the various isoforms of PDGF resulting from post-translational processing, including endoproteolytic cleavage of the PDGF-B chain between Arg-32 and Thr-33. Prior art teaches that structural isoforms resulting from post-translational endoproteolytic cleavage could have varying degrees of biological activity, and hence can effect the overall biological activity of bulk drug substance produced by fermentation.
Methods are needed to separate and quantify the structural isoforms of recombinantly produced PDGF such that their biological activities can be compared. These methods would be useful in preparing bulk drug substances consisting of pure isoforms.